Furthermore, factors associated with QFT-G results in patients with active TB were assessed.In settings with low background prevalence of tuberculosis (TB) infection, interferon-γ release assays (IGRA) could be useful for diagnosing active TB. This study aims to evaluate the performance of QuantiFERONFrom patients investigated for clinically suspected active TB, blood was obtained for QFT-G testing, in addition to routine investigations. Positive (PPV) and negative (NPV) predictive values for QFT-G were calculated, comparing patients with confirmed TB and those with other final diagnoses. QFT-G results in TB patients originating from countries with intermediate or high TB incidence were compared with QFT-G results from a control group of recently arrived asymptomatic immigrants from high-incidence countries. Factors associated with QFT-G outcome in patients with confirmed TB were assessed.9/L vs. 8.8 × 109/L; P < 0.001) and a higher median body mass index  as compared to QFT-G-negative TB patients.Among 141 patients, 41/70 (58.6%) with confirmed TB had a positive QFT-G test, compared to 16/71 (22.6%) patients with other final diagnoses, resulting in overall PPV of 71.9% and NPV of 67.6%. For patients with pulmonary disease, PPV and NPV were 61.1% and 67.7%, respectively, and 90.5% and 66.7% for subjects with extrapulmonary manifestations. Comparing patients from high-incidence countries with controls yielded a PPV for active TB of 76.7%, and a NPV of 82.7%. Patients with confirmed TB and positive QFT-G results were characterized by a lower median peripheral white blood cell count (5.9 × 10The overall PPV and NPV of QFT-G for identifying active TB were unsatisfactory, especially for pulmonary disease. Thus, the usefulness of QFT-G for this purpose is questionable. However, a high PPV was observed for extrapulmonary TB and QFT-G might be considered in the diagnostic process in this situation. The PPV and NPV for identifying active TB among persons originating from regions with high-and intermediate TB incidence was similar to that observed in subjects originating in the low-incidence region. TB (Oxford Immunotec Ltd.) is an assay that enumerates TB specific T-lymphocytes by Elispot technique [®-TB and QuantiFERON®-TB Gold , are based on ELISA-techniques that measure γ-interferon released by TB specific T-lymphocytes [Even in low prevalence regions, TB is a common differential diagnosis in infectious disease practice, and different manifestations of TB may be encountered in all medical disciplines. Whereas most cases of pulmonary tuberculosis can be easily identified by detection of bacteria in airway secretions, the diagnosis of extrapulmonary TB is often difficult to establish. Recently, new diagnostic kits for tuberculosis infection, known as interferon-gamma release assays (IGRA:s), have become commercially available. T-SPOTechnique , whereasphocytes . Both ofphocytes -6, but tphocytes , such asphocytes ,9. In thLike TST, IGRA:s cannot differentiate between active and latent TB. A number of studies examining IGRA results in peripheral blood from patients with active TB has been published, and have found surprisingly low sensitivity -13. ThusSweden has a low incidence of TB (5.5 per 100 000 inhabitants in 2007) . In thisThe principal objective of this study was to define the usefulness of QFT-G in the diagnostic procedure for suspected active tuberculosis among patients in Southern Sweden by calculating PPV and NPV for QFT-G. In order to assess the performance among persons originating from high-prevalence areas, a group of healthy recently arrived immigrants from such countries were compared to those in patients with similar origin. We also aimed to identify factors associated with QFT-G results in patients with confirmed active TB.This study was conducted in Malmö and Lund in Southern Sweden (population ca 400 000). In this area, about 40 cases of TB are reported annually. Study subjects belonged to two categories: case patients with suspected tuberculosis, and healthy immigrants who had recently arrived in Sweden from countries with a high prevalence of TB.Patients were recruited consecutively from the departments of infectious and pulmonary diseases in the university hospitals in Malmö and Lund between September 2004 and February 2007. Inclusion criteria were: age over 15 years, written informed consent, and ongoing investigation for symptoms or signs for which TB was considered as a differential diagnosis. Medical investigation typically included microbiological testing from affected organs, radiological investigations and histopathological studies, according to disease manifestations. Patients were excluded if TB treatment had been initiated more than 1 week prior to study entry, or if TB investigations were done as part of a routine diagnostic package with no definite clinical suspicion of TB . For patients with suspected TB, an interview and record-based questionnaire covering general medical history, risk factors for exposure to TB and details concerning ongoing disease and results of medical investigations was completed by either a physician or a specialized nurse. TST was recommended, but not required, for study inclusion.Since around 80 percent of all TB cases in the study area occur among immigrants from countries with high or intermediate TB incidence  we choseMycobacterium tuberculosis complex from a clinical specimen , or as a patient with tuberculosis clinically suspected by an expert physician, and responding to anti-tuberculosis treatment[A TB case was defined as an individual with symptoms and signs compatible with TB and isolation of, or detection of nucleic acid from, treatment.®-TB Gold test . Samples were transported to the laboratory within 4 hours and incubation with antigens was initiated within 7 hours after collection. Antigens provided in the QFT-G test kit were the 6 kDa early secreted antigenic target (ESAT-6) and culture filtrate protein 10 (CFP-10); in addition to these, TB7.7  and purified protein derivative  were used for incubation experiments. After 16 – 24 hours of incubation at 37°C in 5% CO2, supernatants were collected and frozen at -20°C until analysis. Concentrations of interferon-gamma (INF-γ) in 50 μL of plasma from whole-blood stimulated with the antigens, mitogen (positive control) or saline (negative control) were measured using the ELISA kit provided with the QFT-G package. A positive result was defined as a concentration of INF-γ of ≥0.35 IU/mL after subtraction of the INF-γ concentration from the negative control in at least 1 of the specific antigens ESAT-6, CFP-10 or TB7.7. The INF-γ concentration in the antigen stimulated wells had to be at least half of the concentration of the negative control for the result to be valid. If the mitogen response minus the negative control was < 0.5 the result was defined as indeterminate.Whole blood was collected in heparin-containing tubes for the QuantiFERONThe results of the QFT-G tests were not available to the physicians in charge of the patients, and thus did not influence the diagnostic procedure.TST was done by intradermal injection of 0.1 ml (2 TU) PPD . Skin induration was measured after 72 hours and was interpreted according to the recommendations by the manufacturer. In most cases TST was done prior to QFT-G or at the same time.Positive (PPV) and negative (NPV) predictive values of the QFT-G test among patients were calculated using a final TB diagnosis as true positive and other final diagnoses as true negative outcome. To facilitate comparisons with other studies, calculations of sensitivity and specificity for the diagnosis of active TB were also performed. McNemar test was used to test for overall accuracy. Patients with indeterminate QFT-G results were excluded from the analysis.Patients originating from high and intermediate prevalence regions were compared with healthy immigrant controls to determine PPV and NPV for active TB of both QFT-G and TST in the immigrant population. In patients with confirmed TB, factors associated with positive or negative QFT-G result were first determined by univariate regression analysis. In order to control for possible confounding factors, variables were then put into a multiple regression model.For dichotomous variables an odds ratio was calculated. Differences in odds ratio with a p-value <.05 was considered significant. Odds ratios were defined as the exponent of the estimated beta for dichotomous variables. When comparing means for continuous variables the Student's t-test was used for variables with normal distribution. For other variables the Mann-Whitney's non-parametric test was used.The study procedure was approved by the ethical vetting board, Lund University, Sweden.In 70 of 141 patients, the diagnosis of TB was established, whereas 71 patients received another final diagnosis . Patients with TB were also younger than those with another final diagnosis . Other parameters analyzed did not differ significantly between patients with confirmed TB and non-TB cases.Patients originating from countries with an estimated TB-incidence of more than 20 new cases per 100 000 inhabitants according to the World Health Organization (WHO)[Results of QFT-G for respective patient categories are presented in Table P = 0.268).For the whole group of patients, the PPV and NPV of QFT-G was 71.9% and 67.6%, respectively. The sensitivity and specificity for QFT-G was 63.1% and 75.8% respectively .Among 104 patients with pulmonary disease manifestations, QFT-G was positive in 22 of 44 TB-cases and in 14 out of 60 non-TB patients, .Thirty-seven patients had suspected extrapulmonary TB. Out of these, 21 had a positive QFT-G test, 12 had a negative test and in 4 subjects the QFT-G result was indeterminate .P < 0.001). The mean age of the control subjects was lower than that of the immigrant patients  and the male/female ratio was higher (63/35 vs. 41/42). The most common countries of origin were Iraq (58), the Palestine (11) and Somalia (7) for the immigrant controls, and Serbia (9), Somalia (9), Bosnia-Herzegovina (8), Iraq (8) and Afghanistan (8) for the immigrant patients.The TB incidence in the country of origin was lower among the 98 immigrant controls than for the 52 patients who originated from high and intermediate prevalence regions  had positive QFT-G results. QFT-G results could be determined in 96 of 98 controls. Among these, 10 (10.4%) had a positive result. In control subjects with TST results, the induration was ≥10 mm in 39 of 85 (45.9%) persons.P = 0.327) and for TST 91.2% and 54.1% respectively (McNemar test: P < 0.001).When comparing subjects from high-prevalence countries, the PPV for a positive QFT-G in patients with active TB was 76.7%, and the NPV was 82.7%. The corresponding PPV and NPV for a TST with cut off of 10 mm was 44.3% and 93.9% respectively (Table P < 0.001). The difference between women and men was borderline significant, with a higher likelihood of positive results in women .To further explore factors that may influence the QFT-G results in TB-patients, various parameters were tested. The findings are divided with regard to the variable's quality  and are presented in Table 2) for QFT-G positive TB patients was 22.7 compared to 20.7 for QFT-G negative subjects (Student's t-test: P = 0.043). Patients with a positive QFT-G test had a significantly lower peripheral white blood cell count (WBC) than QFT-G negative subjects . In patients with TST results, the skin induration was significantly larger in QFT-G positive individuals .Mean score of body mass index . In TB patients there was a significant, negative correlation between WBC and QFT-PPD .The antigens added for incubation experiments, TB7.7 and PPD, did not show a significant effect on the results. Six positive QFT-G tests based on TB7.7 only were noted, 2 each in the TB patient group, the non-TB patient group and the group of healthy immigrants. QFT-PPD was used for experiments only and did not give rise to positive QFT-G results alone. The correlation between QFT-PPD and TST was 0.232  . The ass®-TB Gold, with the addition of another MTB specific antigen (TB7.7) apart from those included in the commercial kit. However, the inclusion of this antigen did not appear to affect test outcome. In this study, we have not compared the two different IGRA test methods. Although comparisons between QFT-G and T-SPOT®. TB suggest that the latter may be more sensitive for diagnosing latent TB, the significance of this difference in clinical practice is not clear. Furthermore, QFT has the advantage of being easier and less expensive to perform than T-SPOT®. TB testing. Both IGRA test methods have superior specificity for TB compared to TST, but sensitivity is probably lower (depending on the TST cut-off chosen to define TB infection). However, in analogy with QFT-G, false negative TST reactions are common in severe forms of TB [When assessing the usefulness of IGRA testing for detecting active TB, the immunological profile of the studied patients should thus be taken into account. Our results may not be applicable for HIV-positive patients. Furthermore, several IGRA techniques have been developed, and in a meta-analysis of commercially available IGRA:s differences with regard to sensitivity and applicability have been found ,16. We ums of TB .M. tuberculosis.An important finding from our study is that QFT-G had similar positive and negative predictive values for active TB among native Swedes and immigrants from high-prevalence countries – a group that presently constitutes the majority of TB patients in Sweden as well as in most other European countries. This is somewhat surprising in view of the limited sensitivity of QFT-G for active TB, and the presumed high prevalence of latent TB among the healthy immigrant controls . With regard to the fact that the incidence of TB in the countries of origin among immigrant patients was higher than in controls, it may be anticipated that the exposure to TB was different between these groups. Still, this observation raises further questions regarding the significance of QFT-G and TST for identification of persons infected with The overall usefulness of QFT-G for identifying active TB was limited among patients investigated for suspected TB in a typical setting in a North-European low-prevalence region. Poor performance was more common in patients with pulmonary disease, whereas the PPV for QFT-G was satisfactory among patients with extrapulmonary disease manifestations. Since TB is more difficult to diagnose in such cases, we suggest that it may be reasonable to consider QFT-G for screening patients with suspected extrapulmonary TB. Furthermore, the PPV and NPV of QFT-G for detection of actitve TB among immigrants from high and intermediate prevalence regions seem to be similar to that observed in non-immigrants.The authors declare that they have no competing interests.NW, PB and HM were responsible for planning the study, analyzing the results and drafting the manuscript. HM designed the antigen panel used for QFT-G testing. NW, AN and PB collected the study material and coordinated the study. All authors have read and approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/105/prepub


Long-term potentiation (LTP) and long-term depression (LTD) play important roles in mediating activity-dependent changes in synaptic transmission and are believed to be crucial mechanisms underlying learning and cortical plasticity. In human subjects, however, the lack of adequate input stimuli for the induction of LTP and LTD makes it difficult to study directly the impact of such protocols on behavior.2 on the tip of the index finger. As assessed by 2-point discrimination, we demonstrate that 20 minutes of t-HFS improved tactile discrimination, while t-LFS impaired performance. T-HFS-effects were stable for at least 24 hours whereas t-LFS-induced changes recovered faster. While t-HFS changes were spatially very specific with no changes on the neighboring fingers, impaired tactile performance after t-LFS was also observed on the right middle-finger. A central finding was that for both t-LFS and t-HFS perceptual changes were dependent on the size of the stimulated skin area. No changes were observed when the stimulated area was very small (< 1 mm2) indicating special requirements for spatial summation.Using tactile high- and low-frequency stimulation protocols in humans, we explored the potential of such protocols for the induction of perceptual changes. We delivered tactile high-frequency and low-frequency stimuli  to skin sites of approximately 50 mmOur results demonstrate differential effects of such protocols in a frequency specific manner that might be related to LTP- and LTD-like changes in human subjects. Since its discovery in the early 1970s, long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission had been suggested to be crucial factors for activity-dependent changes in the strength of synaptic connections and efficiency of synaptic signal transduction . DespiteAccordingly, bidirectional synaptic modifications such as LTP and LTD are believed to be crucial mechanisms underlying learning-induced cortical plasticity. However, there is an on-going debate how synaptic plasticity links to systemically observable changes such as cortical map reorganization, and changes in behavior and perception. In human subjects, it is difficult to study the outcome of synaptic modifications on behavioral changes induced by stimuli that drive LTP or LTD-like processes in vivo. Recently, evidence for the functional relevance of such stimulation protocols has been provided in human pain perception. High-frequency stimulation of cutaneous afferents of the proximal forearm resulted in a long-lasting increase in perceived pain to electrical test stimuli. On the other hand, low-frequency stimulation decreased the individual pain perception .In previous studies we took advantage of a stimulation protocol developed in our group that allows for full control of the spatio-temporal pattern of tactile stimuli. In this protocol, we stimulated the tip of the index finger with irregular, Poisson-distributed trains with an average frequency of 1 Hz , which led to improvement of tactile perceptual performance of the index finger in parallel to expansion of cortical representation of the fingers -13. PlasTo evoke plastic processes in the cortical representations of the stimulated skin sites, in the present study we delivered regular trains of either high-frequency (HFS) or low-frequency (LFS) tactile stimuli to the tip of the index finger by means of small stimulation devices (for review see ). We hypAs an output measure we used tactile spatial 2-point discrimination performance as a marker for its perceptual relevance. Here we demonstrate that brief periods (20 minutes) of intermittent high-frequency (HFS @ 20 Hz) and continuous low-frequency (LFS @ 1 Hz) protocols of tactile stimulation applied to the index-finger (d2) of the right hand evokes significant and long-lasting changes in tactile discrimination behavior. = 0.759; p = 0.524; n = 14). After applying t-HFS for 20 minutes discrimination thresholds on the right d2 were reduced. Discrimination thresholds were 1.60 ± 0.08 mm before t-HFS and 1.34 ± 0.09 mm after stimulation resulting in an average gain of tactile performance of 0.25 ± 0.04 mm  = 33.712; p < 0.0001; see Fig.  = 5.771; p = 0.032). Re-testing one week after t-HFS revealed that discrimination thresholds recovered to conditions found prior to t-HFS  = 2.577; p = 0.132; see Fig. During the four initial training sessions s1–s4 = pre) all subjects achieved a stable baseline of discrimination performance on their right d2 as estimated by repeated measures ANOVA with factor SESSION  = 13.506; p = 0.002), only little changes could be observed for all other fingers. We found alterations of thresholds of 0.07 ± 0.14 mm (-3.69 ± 9.70%) for the thumb (d1), 0.07 ± 0.17 mm (-2.43 ± 6.91%) for the middle-finger (d3), 0.07 ± 0.07 mm (-2.15 ± 2.63%) for the ring-finger (d4) and of -0.08 ± 0.09 mm (3.54 ± 3.30%) for the little-finger (d5). According to rmANOVA, none of these changes were significant. These findings demonstrate a clear absence of effects on other fingers than the stimulated d2, which indicates a substantial local specificity of the tactile HFS-induced changes  after t-HFS  = 0.986; p = 0.432; n = 5). Stimulating the right index-finger (d2) using 20 minutes of small-field t-HFS resulted in no comparable changes of tactile discrimination thresholds  = 2.283; p = 0.205; n = 5, see Fig. Prior to small-field t-HFS, all subjects achieved a stable baseline performance  = 0.052; p = 0.984, see Fig.  = 10.608; p = 0.007, see Fig.  = 1.209; p = 0.293) implying that the t-LFS-induced impairment was less persistent than the improvement observed after t-HFS. Additional measurements one week after LFS application showed that discrimination thresholds remained unchanged as compared to baseline conditions  F = 0.958; p = 0.347, see Fig. We assessed the outcome of large-field t-LFS on 2-point discrimination in 13 right-handed subjects. All subjects achieved a stable baseline performance, as estimated from repeated assessment of thresholds over 4 consecutive sessions  = 6.985; p = 0.018), but also on the adjacent finger d3  = 6.963; p = 0.019, see Fig. Similar to the experiments described for t-HFS, we explored whether besides d2 the other fingers of the right hand were also affected by t-LFS. Baseline discrimination performance (d1–d5) on the right hand n = 16) before t-LFS did not differ from the subpopulation that was used to study the local specificity of t-HFS-induced changes except for d3 (paired t-test: p = 0.032). In contrast to t-HFS, brief periods of t-LFS (20 min) resulted in changes of tactile discrimination thresholds not only on the right stimulated d2 (rmANOVA with factor SESSION (pre vs. post) F before t = 0.971; p = 0.438; n = 5). Moreover, baseline performance did not differ between the groups tested with either small-field t-LFS or small-field t-HFS  = 0.459; p = 0.517). Applying 20 minutes of small-field t-LFS resulted in no changes of tactile discrimination thresholds  = 0.03; p = 0.957, see Fig. Prior to small-field t-LFS, all subjects achieved a stable baseline performance (rmANOVA with factor SESSION: F2) implying the need for spatial summation.The psychophysical results shown provide the first evidence for the effectiveness of t-HFS and t-LFS protocols to evoke tactile performance changes in human subjects. We found that brief periods of intermittent high-frequency tactile stimulation (t-HFS @ 20 Hz) applied to the skin of the right d2 induced a lowering of 2-point discrimination thresholds on that finger. Most interestingly, 24 hrs after t-HFS 2-point discrimination thresholds on the right d2 were still lowered in comparison to baseline indicating that brief periods of t-HFS can induce relatively long-lasting perceptual changes. These changes were spatially very specific as we did not observe any changes on the neighboring fingers. In contrast to t-HFS, 20 minutes of continuous low-frequency stimulation (t-LFS @ 1 Hz) led to a decline in tactile discrimination performance on the right stimulated d2. However, these changes were less lasting and recovered to baseline after 24 hours. T-LFS effects differed further in terms of spatial specificity. Impaired tactile performance was also observed on the right middle-finger. A central finding was that for both t-LFS and t-HFS perceptual changes were dependent on the size of the stimulated skin area. No changes at all were observed when the area was very limited , a larger effect on discrimination might have been expected, which was, however, not the case. Instead, the clear lack of effects evoked by small-field stimulation in both the t-HFS and t-LFS condition implies that certain spatial summation requirements need to be fulfilled in order to drive behavioral changes. Because small-field stimulation is clearly above threshold for evoking distinct sensation felt by the subjects, it is reasonable to assume that stimulation of this type also activates neurons located in the finger representation of somatosensory cortex. However, because of its limited size this cortical activation is not sufficient to overcome the threshold mechanisms to initiate plastic processes.In all cases the protocols we here employed were local, restricting stimulation to the tip of the index finger, which was effective to alter 2-point discrimination on the skin regions stimulated. Therefore, another aim of the present study was to test whether tactile stimulation of the index finger also effects 2-point discrimination of the neighboring fingers. For t-HFS no changes could be detected on other than the stimulated finger which indicates a substantial spatial specificity with no transfer and spread on neighboring fingers. For t-LFS, the situation was quite different, as a significant impairment of threshold was not only observed for the stimulated index finger, but also for the middle finger. After t-LFS, thresholds were also higher for d1 and d4, although these changes were not significant Fig. . AccordiA number of studies have provided evidence according to which prolonged, but unattended stimulation is ineffective to drive plastic changes -26. On tThe fact that in all cases under all conditions tested the discrimination thresholds on d5 remained unaffected can be taken as an argument against nonspecific side effects such as overstimulation, habituation, and alterations in local blood circulation or finger temperature. Influences of this type seem also very unlikely to play a role because of the opposite direction of effects evoked by using different stimulation frequencies. Also, systematic shifts in attention dependent of stimulation frequency would have likely impacted to similar extents the tactile performance of all fingers. According to recent unpublished data from our group, attending the coactivation stimuli during coactivation or being distracted from them had no effect on the overall outcome of coactivation. Therefore, the differential effects in 2-point discrimination induced by tactile high- and low-frequency stimulation are unlikely to be mediated by attentional processes. Interestingly, stimulation that effectively controls the timing of applied stimuli, as it is the case in peripheral tactile stimulation, might short cut the role of attention by producing the same conditions as attention, such as synchronous firing or increased probability of firing in specific temporal order among groups of neurons ,29.In summary, the reported changes in 2-point discrimination in human subjects induced by either t-HFS or t-LFS provided first evidence for the perceptual relevance of stimulation protocols resembling those used in cellular LTP and LTD studies. Conceivably, further extension of these approaches will be beneficial for the understanding of possible mechanisms underlying tactile performance changes, and for developing novel strategies in neurorehabilitation.We tested a total number of 70 healthy subjects between 20 and 36 years (mean age 24.62 ± 4.42 years (SD)) in a psychophysical task using tactile spatial 2-point discrimination as a marker for alterations in human tactile perception. Subjects were randomly allocated to 2 experimental conditions (high-frequency tactile stimulation (t-HFS), n = 36; low-frequency tactile stimulation (t-LFS), n = 34): For an overview of subject's assignment to different tests see Table Tactile 2-point discrimination on the fingers was assessed using the method of constant stimuli as described previously . t-HFS and t-LFS consisted of brief rectangular pulses (10 ms duration) of tactile stimuli that were applied to the distal phalanx of the right index-finger (d2) to the position where 2-point discrimination was performed. Pulse trains required to drive the stimulators were stored digitally as TTL pulses and played back via MP3 player allowing unrestricted mobility of the subjects during the stimulation period. For t-HFS, stimulation trains consisted of 20 single pulses within 1 s with an inter-train interval of 5 s. T-LFS was applied continuously at a stimulation frequency of 1 Hz. Duration of t-HFS or t-LFS application was 20 min, resulting in a total number of 4000 pulses for t-HFS and 1200 pulses for t-LFS. In all cases, subjects were instructed not to attend the stimulation but to resume their daily routine. Although the spectral power of the single pulses was "high-frequency" (pulse duration 10 ms), we use the term "low" or "high-frequency" stimulation to describe the number of pulse per second (pps) in terms of Hz to connect to the terms used in cellular studies of brain plasticity.2 on the tip of the IF was stimulated. We called this protocol large-field stimulation. In the other version a very restricted (point-like) skin area of only 0.8 mm2 was stimulated, which is termed small-field stimulation. For application of the large-field stimulation, we used a device consisting of a solenoid connected to a flexible membrane with a diameter of 8 mm, which was fixed to the tip of d2. Laser vibrometer measurements revealed that the actual amplitude of the membrane movement was about 100 microns. For application of the small-field stimulation, we used a tiny stimulator consisting of a small needle probe (diameter 0.5 mm) that was also taped to the tip of the right d2. The probe was connected to the switch of a relay that changed its vertical position whenever a voltage of 5 V was applied. Changes in voltage were controlled via a small electrical amplifier that was driven by the stimulation pulses from the MP3 player. Amplitude of the probe movement was in the range of 200 microns. By comparing the effects induced by either large- or small field stimulation information about spatial summations processes can be obtained. Sensations elicited by the stimulation were very soft but perceptible resembling a very light touch on the stimulated skin site. In order to study the spatial selectivity of t-HFS/t-LFS-induced perceptual effects outside the stimulated d2, we studied alterations in tactile performance on all other fingers of the right hand in two additional groups (Table For both t-HFS and t-LFS we used two variants that differed with respect to the skin-size stimulated. In the standard protocol a skin area of approximately 50 mmThe experiment consisted of two different components: (1) the measurement of 2-point discrimination thresholds; (2) tactile HFS or LFS of 20 minutes duration each to induce perceptual changes on the right d2. Prior to t-HFS/t-LFS, discrimination thresholds were tested on 4 consecutive sessions (session (s) 1–4) on one day in order to obtain a stable baseline performance. Testing 2-point discrimination for each session lasted for approx. 5 minutes and was separated by 2 minutes Fig. . In expePsychophysical data were statistically analyzed using repeated measures ANOVA (rmANOVA) with factor SESSION or univariate ANOVA (uANOVA) with factor FINGER or GROUP as well as Student's paired t-tests, post-hoc analysis corrected for multiple comparisons (Bonferroni) and correlation analysis (Pearson correlation coefficient). Data were normally distributed as evaluated by Kolmogorov-Smirnov tests. Inspection of the data showed that false alarms were zero under each condition. All data are expressed as mean ± s.e.m. (standard error of mean).PR conceived of the study, carried out parts of the psychophysical measurements, performed the statistical analyses, and drafted the manuscript. BB and SF carried out parts of the psychophysical experiments. BB also helped to draft the manuscript. TK participated in the study design. HRD participated in the study design and coordinated and helped to draft the manuscript. All authors read and approved the final manuscript.


Metallothionein (MT) isoforms I + II are polypeptides with potent antioxidative and anti-inflammatory properties. In healthy kidneys, MT-I+II have been described as intracellular proteins of proximal tubular cells. The aim of the present study was to investigate whether the renal MT-I+II expression profile is altered during lupus nephritis.Immunohistochemistry was performed on renal biopsies from 37 patients with lupus nephritis. Four specimens of healthy renal tissue served as controls. Clinicopathological correlation studies and renal survival analyses were performed by means of standard statistical methods.P = 0.006). The tubular MT score, defined as the median number of proximal tubules displaying this MT expression pattern per high-power microscope field (40× magnification), was positively correlated to the creatinine clearance in the lupus nephritis cohort (P = 0.01). Furthermore, a tubular MT score below the median value of the cohort emerged as a significant predictor of a poor renal outcome in renal survival analyses. Thus, patients with a tubular MT score < 1.0 had a 6.2-times higher risk of developing end-stage renal disease than patients with a tubular MT score ≥ 1.0 (P = 0.03).Proximal tubules displaying epithelial cell MT-I+II depletion in combination with luminal MT-I+II expression were observed in 31 out of 37 of the lupus nephritis specimens, but not in any of the control sections (Lupus nephritis is associated with significant alterations in renal MT-I+II expression. Our data indicate that important prognostic information can be deduced from the renal MT-I+II expression profile in systemic lupus erythematosus patients with nephritis. Systemic lupus erythematosus (SLE) is an immune-inflammatory disorder characterized by multiorgan involvement and a chronic relapsing course. Nephritis is a common and serious manifestation of SLE. More than one-half of all SLE patients develop nephritis during their course of illness, and 10% to 25% of these patients progress to end-stage renal disease (ESRD) despite immunosuppressive treatment . A centrMetallothioneins (MTs) are nonenzymatic polypeptides of 6 to 7 kDa that bind heavy metals with high affinity and possess a range of anti-inflammatory properties . In addiThe aim of the present study was to investigate whether the pathogenesis of lupus nephritis involves an altered pattern of renal MT-I+II expression. To our knowledge, this question has not been addressed before. We demonstrate that lupus nephritis is associated with significant alterations in the tubular MT-I+II expression profile. Furthermore, our data indicate that important prognostic information can be deduced from the renal MT-I+II expression pattern in patients with lupus nephritis.Formalin-fixed, paraffin-embedded renal biopsies were obtained from patients of a previously described historic lupus nephritis cohort . All patThe following data were registered at the time of biopsy for all SLE patients: sex, age at time of biopsy, age at time of SLE diagnosis, date of first renal biopsy, date of onset of nephritis symptoms (defined as the date of the first observation of persistent proteinuria (> 0.5 g/day), hematuria, and/or cellular casts), serum creatinine, creatinine clearance, serum albumin, blood pressure, level of 24-hour urinary protein excretion, a clinical disease activity score  and classified according to the World Health Organization (WHO) classification criteria for lupus nephritis . Activit2O2 in 3.3-diaminobenzidine-tetrahydrochloride (DAB)/Tris-buffered saline  for 10 minutes at room temperature. To evaluate the extent of nonspecific binding in the immunohistochemical experiments, control sections were incubated in the absence of primary antibody.Paraffin-embedded renal biopsies were cut into 5 μm-thick sections and were processed for immunohistochemistry as previously described ,20. In bTo obtain an overall impression of the MT-I+II expression patterns, anti-MT-I+II-stained sections were first screened by two investigators (MF and MP). In the healthy kidney specimens, a strong MT-I+II staining reaction was observed in the cytoplasm of proximal tubular cells. Furthermore, cytoplasmic MT-I+II staining was seen in the epithelial cells of some collecting tubules as described below. In the lupus nephritis specimens, MT-I+II staining was also found in the proximal renal tubules. In a large proportion of these specimens, however, some proximal tubules displayed intense luminal MT-I+II reactivity in combination with absent or significantly reduced epithelial cell staining. The number of cross-sectioned proximal tubules displaying the latter pattern of anti-MT-I+II reactivity was determined in a sequence of 3 to 60 consecutive high power fields (40× magnification), depending on the size of the individual biopsy. The only field adjustments were made to avoid large vessels and glomeruli.For semiquantitative analyses, each cross-sectioned proximal tubule displaying significant luminal MT-I+II reactivity in combination with reduced/absent staining of the lining epithelial cells was scored one, while cross-sectioned proximal tubules in which the MT-I+II staining was observed exclusively/predominantly in the cytoplasm of the epithelial cells were scored zero. As a conservative approach, tubules that did not clearly display one of these morphologies were also scored zero. The tubular MT score of the individual biopsy specimens was defined as the median score obtained according to this scoring system per visual field at 40× magnification.Most of the lupus nephritis biopsies consisted mainly of cortical renal tissue. Consequently, the MT-I+II expression in collecting tubules could not be systematically examined in these specimens. In all biopsies, glomerular cross-sections were also evaluated for MT-I+II reactivity, and the median number of MT-I+II-positive cells was determined.All biopsies were scored by the same investigator (MF) and were analyzed without knowledge of clinical or standard histopathological data. The scorings were subsequently validated by another investigator (CBA).Sections were examined on an Axioplan plus light microscope . Images were recorded using a digital camera .The cumulative incidence of ESRD was calculated using life tables and the Kaplan–Meier method. The equality of cumulative incidence curves was tested by the log-rank test. Event rates were related to the total number of person-years of observation, and rate ratios of event rates were calculated in stratified analyses. Comparison of continuous data was performed using the Mann–Whitney rank-sum test. Spearman's rank correlation test was used in correlation studies.P < 0.05 (two-tailed) was considered statistically significant. The statistical analyses were performed on a computer using SPSS version 9.0 for Windows .In all analyses, The present study was approved by the local ethics committee  and by the Danish Data Protection Agency .Clinical data of the 37 SLE patients are summarized in Table P < 0.0001). In contrast, the median chronicity index score was not significantly higher in patients with WHO class IV nephritis (median chronicity index score = 1.0 (range = 0 to 9)) than in patients displaying other renal histopathologies (median chronicity index score = 0.0 (range = 0 to 3); P = 0.21).Four patients (11%) presented with WHO class II glomerulonephritis, three patients (8%) with WHO class III glomerulonephritis, 27 patients (73%) displayed WHO class IV histopathology, and three patients were diagnosed with WHO class V glomerulonephritis (8%). The median histological activity index score in the cohort was 5.0 (range = 0 to 14), while the median chronicity index score was 1.0 (range = 0 to 9). The median activity index score was significantly higher for patients presenting with WHO class IV glomerulonephritis (median activity index score = 7.0 (range = 2 to 14)) than for patients with other WHO classes of nephritis (median activity index score = 1.5 (range = 0 to 4); Anti-MT-I+II staining of control specimens confirmed that MT-I+II are expressed in the cytoplasm of proximal tubular cells under normal conditions Figure  13,14].,14.13,14Three of the four control sections contained enough medullary renal tissue for an examination of medullary structures to be performed. As a novel finding ,14, we dIn six out of the 37 lupus nephritis specimens, intense cytoplasmic MT-I+II staining was observed in the epithelial cells lining the proximal tubules Figure . The MT-Only nine (24%) of the lupus nephritis biopsies contained significant amounts of medullary renal tissue. In these specimens, collecting tubules displayed the same epithelial MT-I+II expression pattern as observed in the control biopsies (data not shown). Glomeruli and interstitial structures were MT-I+II-negative in all lupus nephritis specimens Figure  to 1f.To evaluate whether similar changes in renal MT-I+II expression occur in the setting of other inflammatory nephropathies, we finally studied the tubular MT-I+II expression pattern in biopsies from five patients with AASV and glomerulonephritis. In four out of five of these biopsies, we observed the occasional presence of tubules displaying the same alterations in MT-I+II expression as described above Figure .P = 0.006) and in the AASV biopsies (median tubular MT score of the five AASV specimens = 0.16 (range = 0.0 to 0.27); P = 0.04). Among the SLE patients, the median tubular MT score tended to be lower for patients with WHO class IV nephritis than for patients presenting with other WHO histopathologies (median tubular MT score for 27 patients with WHO class IV nephritis = 0.8 (range = 0.0 to 6.8); median tubular MT score for the 10 other patients = 1.4 (range = 0.1 to 3.6)). This difference was not, however, statistically significant (P = 0.07).The median tubular MT score was significantly higher in the lupus nephritis specimens (median tubular MT score of the 37 lupus nephritis biopsies = 1.0 (range = 0.0 to 6.8)) than in the control specimens (median tubular MT score of the four control specimens = 0.0 (range = 0); n = 6) differed from patients with tubular MT score > 0 (n = 31) with regards to baseline levels of selected variables reflecting renal function, including creatinine clearance, level of 24-hour urinary protein excretion, serum albumin, activity index score, chronicity index score, and blood pressure  ligand 16 ,32. Furtper se.Intriguingly, our analyses demonstrate that proximal tubules do not always display intraluminal MT-I+II immunostaining during lupus nephritis, suggesting that this alteration in renal MT distribution requires or can be blocked by as yet undefined stimuli. To this end, it could be hypothesized that proximal tubular cells secrete MTs in response to the presence of harmful substances delivered to the tubular lumen from the inflamed glomerulus, but that this secretion can be inhibited by other intracellular or extracellular signals associated with renal inflammation. Although the present study was not designed to investigate the extent to which similar changes in renal MT expression occur during other inflammatory nephropathies, our analyses indicate that the same morphological tubular alterations are seen in AASV glomerulonephritis. This observation suggests that the mechanisms responsible for inducing the observed alterations in the tubular MT expression profile are not specific for lupus nephritis, but are probably evoked by renal inflammation A range of histological features has been shown to influence the prognosis in lupus nephritis. Frequently reported histological predictors of a poor renal outcome include WHO class IV histopathology ,33,34, hFurthermore, our study raises a number of new questions. Based on the known functional repertoire of MT-I+II, it is tempting to speculate that MT-I+II protect the proximal tubular epithelium from reactive oxygen species and other cytotoxic substances after secretion to the tubular lumen as outlined above. Nevertheless, the mechanisms by which these MTs provide renoprotection during lupus nephritis remain to be defined. It also remains to be clarified to what extent the observed changes in tubular MT-I+II expression are evoked in the setting of other inflammatory nephropathies. Finally, from a therapeutic perspective it would be of great interest to investigate whether treatment with exogenous MT-I+II could modify the course of lupus nephritis in animal models.Our study provides the first description of renal MT-I+II expression during lupus nephritis. Compared with the MT-I+II expression pattern observed in healthy renal tissue, characteristic alterations in the tubular MT expression profile were found in most of the examined lupus nephritis specimens. In renal survival analyses, these alterations were found to be of prognostic significance.AASV = antineutrophil cytoplasmic autoantibody-associated systemic vasculitis; ESRD = end-stage renal disease; IL = interleukin; MT = metallothionein; SLE = systemic lupus erythematosus; TNF = tumor necrosis factor; WHO = World Health Organization.The authors declare that they have no competing interests.MF, MP, and SJ designed the study. MF analyzed the immunohistochemically stained slides, performed the data analysis, and prepared the manuscript. MP performed the immunohistochemistry assays and contributed to the analysis of the stained slides. CBA contributed to the analysis of the stained slides and validated the tubular MT scorings. HS performed the standard histopathological analyses. SJ provided the clinical data of the cohort and contributed to the data analysis. All authors read and approved the final manuscript.


Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP+ or NAD+ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR.Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in CtXR and CbFDH activity per gram of dry E. coli cell mass (gCDW). The maximum conversion rate (rS) for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/gCDWh), suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH in the presence of the α-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions , we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e.e.) of 97.2% in a yield of 82%. By increasing the substrate concentration to 500 mM, the e.e. could be enhanced to ≅100%, however, at the cost of a 3-fold decreased yield. A recombinant strain of S. cerevisiae converted 100 mM substrate to 45 mM ethyl R-4-cyanomandelate with an e.e. of ≥ 99.9%. Modifications to the recombinant E. coli  and addition of a water immiscible solvent (e.g. hexane or 1-butyl-3-methylimidazolium hexafluorophosphate) were not useful. To enhance the overall capacity for NADH regeneration in the system, we supplemented the original biocatalyst after permeabilisation with also permeabilised E. coli cells that expressed solely CbFDH (410 U/gCDW). The positive effect on yield  caused by a change in the ratio of FDH to XR activity from 2 to 20 was invalidated by a corresponding loss in product enantiomeric purity from 86% to only 71%.Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular E. coli co-expressing CtXR and CbFDH is a powerful and surprisingly robust biocatalyst for the synthesis of ethyl R-4-cyanomandelate in high optical purity and yield. A clear requirement for further optimization of the specific productivity of the biocatalyst is to remove the kinetic bottleneck of NADH regeneration through enhancement (≥ 10-fold) of the intracellular level of FDH activity.A whole-cell system based on  The spatial organisation of the whole-cell system where enzymes and cofactors are encapsulated by the supramolecular structure of the cell membranes potentially improves the efficiency of coenzyme recycling as compared to homogeneous reactors employing "free-floating" biocatalytic components.Enzyme-catalyzed enantioselective reductions of ketones have become quite popular for the production of homochiral alcohols at industrial scale . NAD(P)HEscherichia coli to over-express various synthetically useful ketoreductases to a high level of activity, this organism has become a prime choice for the development of whole-cell bioreduction catalysts. The capabilities of E. coli to provide internal cofactor regeneration are, however, oftentimes limiting overall [+ or NADP+-dependent dehydrogenase is therefore used to couple the biosynthetic reduction of the target ketone with the oxidation of a suitable co-substrate. Currently, oxidation of glucose catalyzed by glucose dehydrogenase (GDH) is most often used for cofactor regeneration [E. coli involves coupled transport and phosphorylation. The resulting glucose 6-phosphate is not a substrate of GDH. To provide glucose efficiently for GDH-catalyzed oxidation, one must therefore make the cell membrane just sufficiently permeable for glucose (but not for coenzyme) or engineer the glucose uptake system [Considering the ability of  overall -9. Co-exneration ,6,9-14. e system -17. Forme system ,12,14,17e system -20. The e system .E. coli cell. It should not by itself or the product generated from it, inhibit or inactivate the synthetic ketoreductase. Oxidation of the co-substrate should be thermodynamically favoured, and the co-product should be easily removed from the reaction mixture. These demands are widely met by the conversion of formate into carbon dioxide catalyzed by formate dehydrogenase (FDH) [+ and can be used in a broad pH range , the development of corresponding whole-cell systems is not advancing as quickly. The relatively low specific activity of CbFDH  ,17,22-266.0–9.0; ). While  is consE. coli BL21 (DE3) through co-expression of the genes encoding Candida tenuis xylose reductase (CtXR) and CbFDH. It was shown in previous work that CtXR promotes the reduction of aromatic α-keto esters with useful efficiency and excellent stereoselectivity [R-4-cyanomandelate was employed to characterize and optimize the biocatalytic performance of the recombinant E. coli strain . We placed the gene encoding CbFDH after the CtXR gene with the aim of specifically enhancing the overproduction of the formate dehydrogenase, as recommended by the supplier Novagen.The vector pETDuet-1-XR_FDH was constructed to enable co-expression of the genes encoding  is abouCtXR and CbFDH in E. coli BL21 (DE3), we compared low (0.25 mM) and high (1.00 mM) IPTG concentrations in combination with short (5 h) and long (20 h) induction times. The specific activity of CtXR in the cell-free extract was not dependent on the IPTG concentration but enhanced from 0.58 U/mg to 0.91 U/mg by using the long induction phase. The specific activity of CbFDH (0.18 U/mg) was insensitive to changes in any of the two cultivation parameters. By way of comparison, the specific activity of CbFDH in the cell extract of E. coli FDH was 1.4 U/mg. The specific activity of CtXR in E. coli XR_FDH compares roughly to one of 0.49 U/mg that is obtained when using the expression vector pBEAct.1i in E. coli BL21 (DE3). The results also show that the activity ratio CtXR/CbFDH in E. coli XR_FDH was ~8-fold higher than expected from the specific activities of the purified enzymes. Therefore, differential overproduction of CtXR and CbFDH to favour formation of the formate dehydrogenase did not occur with the expression construct used. We inspected E. coli XR_FDH under the light microscope and observed that a substantial amount of protein inclusion bodies was formed under induction conditions (data not shown). While intracellular precipitation could clearly account for losses of CbFDH activity, we considered the examination of folding factors for CbFDH in E. coli to be outside the scope of this paper. The level of CbFDH expression using pETDuet-1-XR_FDH was comparable to that seen in previous work where CbFDH and an alcohol dehydrogenase were co-expressed in E. coli BL21 (DE3) from a pACYCDuet-1 vector [In a screening for conditions that are suitable for the simultaneous production of 1 vector . Note thE. coli XR_FDH. To facilitate the comparison of different whole-cell catalysts, we normalized the results using gCDW. The activity of CtXR (using xylose as the substrate) in E. coli XR_FDH was about 7 times the corresponding enzyme activity in S. cerevisiae XR2μ. The levels of the enzyme activities exploited in the regeneration of NADH were similar in E. coli XR_FDH and S. cerevisiae XR2μ . With the assay used [+-dependent aldehyde dehydrogenase (AlDH) activity in S. cerevisiae XR2μ was below detection limit (< 0.2 U/gCDW). The wild-type strain of S. cerevisiae as well as E. coli FDH shows no ethyl 4-cyanobenzoylformate reductase activity in the absence of CtXR overexpression.Table say used , the levrS) by E. coli XR_FDH, S. cerevisiae XR2μ, and the wild-type strain of S. cerevisiae. A low substrate concentration of 10 mM was chosen to decrease toxic effects of ethyl 4-cyanobenzoylformate on the cells (see later). Yeast bioreduction experiments were carried out under anaerobic conditions in the presence of ethanol as co-substrate to promote ADH-catalyzed regeneration of NADH while suppressing at the same time routes that could recycle NADPH. Kratzer et al. [S. cerevisiae XR2μ proceeded under these conditions with the near perfect enantioselectivity of isolated CtXR in analogous conversions. Initial rates (rS) were calculated from linear time courses of substrate conversion in the first ≅ 1 h of reaction, and results are summarized in Table rS-Values for the bacterial strain and S. cerevisiae XR2μ were similar and surpassed that for the wild-type yeast by a factor of 10. The comparison of data in Tables rS values is not reflected clearly in the activity levels for the biosynthetic and NADH-regenerating enzymes that are present in the different cells. However, we must consider that rS is a kinetically complex parameter , the yield from 100 mM ethyl 4-cyanobenzoylformate was 45% (data not shown).Figure E. coli XR_FDH cells, as shown in Figure rS is much larger throughout than the decomposition rate of the substrate. Likewise, unless stabilized against inactivation by the organic substrate, cells cannot be re-used for multiple rounds of batchwise substrate conversion. Feeding the substrate at a moderately toxic concentration [Time courses of reduction of ethyl 4-cyanobenzoylformate were characterized by a sharp decrease in the conversion rate which irrespective of the amount of product already formed under the different experimental conditions, occurred at approximately the same time during the initial phase of the transformation Figure . Chemicantration  and the S. cerevisiae XR2μ was 2.7 h, a ≅ 2-fold increase in enzyme stability as compared to the E. coli XR_FDH system. According to literature [E. coli to the denaturing effects of organic compounds, irrespective of whether they are soluble or present as a second liquid phase. Therefore, this could explain the observed stabilisation of XR. Note that a soluble preparation of XR has a half-life time of 3.7 min when exposed to 100 mM ethyl 4-cyanobenzoylformate, indicating that the whole-cell environment of S. cerevisiae or E. coli generally provides significant protection to the enzyme. However, with a half-life of only 0.8 h, the stability of ADH appeared to determine overall lifetime of the yeast whole-cell catalyst under the conditions used for α-keto ester reduction.The half-life time of XR in terature  baker's P value increases. We therefore selected three common solvents covering a logP range from 0.68 to 3.5  and one ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6)). The BMIMPF6 co-solvent was chosen considering the report of Bräutigam et al. [E. coli XR_FDH (Table S. cerevisiae XR2μ (data not shown) displayed a significantly poorer biocatalytic performance in the presence of co-solvent than in the absence thereof. By way of comparison, the use of BMIMPF6 caused ≅ 13-fold enhancement of space-time-yield in the reductive conversion of a β-keto ester catalyzed by whole cells of E. coli co-expressing a ketoreductase and bacterial FDH [E. coli and S. cerevisiae in the development of whole-cell biocatalysts [We considered that a water-immiscible organic co-solvent could, by lowering the substrate concentration in the aqueous phase which contains the whole-cell catalyst, improve the stability of all critical components in the reaction system for ethyl 4-cyanobenzoylformate reduction. A widely held notion is that the toxicity of organic solvents on whole cells decreases as their logm et al.  where thm et al. ,35,36), rial FDH . Therefoatalysts ,38, therrS can in certain cases be enhanced by making the cell wall more easily permeable for low-molecular weight compounds such as substrates, products and if applied, external NAD(P) cofactors [E. coli XR_FDH in the conversion of ethyl 4-cyanobenzoylformate. Likewise, the addition of NAD+  of ethyl R-4-cyanomandelate from 100 mM substrate was obtained when using aqueous buffer lacking co-solvents and other additives. Note that conversion of the α-keto ester via enzymatic reduction and the competing route of chemical decomposition was complete in all experiments, implying that ≥ 20% of the used substrate was lost to non-enzymatic transformations. The absence of a rate-enhancing effect of external NAD+ may be explicable on account of the total intracellular concentration of NAD(H) which is around 1 mM [Km values of CtXR [CbFDH [+ which are 38 μM and 90 μM, respectively, it seems probable that the levels of reduced and oxidized coenzyme in E. coli are high enough to saturate the coupled enzyme system. Permeabilization of S. cerevisiae XR2μ was not pursued.Evidence from previous studies of ketone reduction by whole-cell biocatalysts suggests that ofactors ,39-43. Fofactors , we seleofactors . Data inAD+ see ,13,23) t,23 trS cund 1 mM . Conside of CtXR  and CbFDE. coli XR_FDH showed a XR to FDH activity ratio of 3:1, which may not be optimal for biocatalytic synthesis. The level of FDH activity in E. coli FDH was 5 times that of E. coli XR_FDH  and the activity of FDH was varied at levels of 3, 9 and 27 U/mL. Both E. coli strains were permeabilized using polymyxin B sulphate, and external NAD+ was also added. Results are summarized in Table rS of E. coli XR_FDH is limited by NADH regeneration capacity. The presence of external NAD+ did not stimulate rS in the two-strain system, indicating that it is availability of NADH, however not that of total coenzyme that restricts the synthesis rate. Using a comparable system where one E. coli strain expressed the synthetic ketoreductase and another GDH for regeneration of NADPH, Xu et al. [E. coli ketoreductase background in the overall conversion of the ketone substrate  of the intracellular level of FDH activity. Because yeast FDHs in their wild-type forms show rather low specific activities [A whole-cell system based on eduction . Ethyl 4tivities ,48), powtivities ,17,22,24+  and NADPH  were obtained from Roth .Ethyl 4-cyanobenzoylformate was purchased at Sigma-Aldrich . Racemic ethyl 4-cyanomandelate was from Synthon Chemicals GmbH & Co. KG . NADH , NADE. coli JM109, E. coli BL21 (DE3), S. cerevisiae CEN.PK 113-7D (MATa MAL2-8c SUC2) (termed S. c. wild-type) and S. cerevisiae CEN.PK 113-5D . Pfu DNA polymerase was from Promega . dNTPs, T4 DNA ligase, and restriction enzymes were from MBI Fermentas . The ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6) was from Sigma-Aldrich (product number 18122). All other chemicals were purchased from Sigma-Aldrich/Fluka  or Roth , and were of the highest purity available.The microorganisms used were CbFDH gene (from strain C. boidinii ATCC 18810) into the plasmid vector pBTac1 (pBTac1-FDH) was described previously [E. coli JM109 harbouring pBTac1-FDH was termed E. coli FDH. The pETDuet-1 vector from Novagen  was used for co-expression of the genes encoding CtXR and CbFDH. This vector contains two multiple cloning sites, each of which is preceded by a T7 promoter/lac operator and an optimal ribosome binding site for high level protein expression [CtXR (from C. tenuis CBS 4435) was amplified from the plasmid pBEAct.1i [Pfu DNA polymerase and the following pair of oligonucleotide primers which provided PagI (compatible ends to NcoI) and HindIII restriction sites.All DNA manipulations and bacterial transformations were carried out according to standard protocols. Cloning of the eviously . The strpression . The vecBEAct.1i  by a PCRTCATGAGCGCAAGTATCC-3',forward primer: 5'- GGTGGTAAGCTTTTAAACGAAGATTGGAATG -3'.reverse primer: 5'- ACCACCNcoI, HindIII), yielding pETDuet-1-XR. The CbFDH gene was likewise amplified from pBTac1-FDH using the oligonucleotide primers listed below.These restriction sites were used subsequently to clone the gene into the first multiple cloning site of pETDuet-1  was transformed with pETDuet-1-XR_FDH using a standard electroporation method. The correct integration of the genes for CtXR and CbFDH and the absence of misincorporations of nucleotides due to DNA polymerase errors were verified by sequencing. The resulting strain was termed E. coli XR_FDH. Construction of the strain S. cerevisiae XR2μ was described previously [S. cerevisiae CEN.PK 113-5D and harbours the yeast 2μ expression plasmid p426GPD that contains the gene for CtXR under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter.The primers provided eviously . The strE. coli strains were grown in 1000 mL baffled shaken flasks containing 200 mL of LB media supplemented with 115 mg/L ampicillin. A Certomat® BS-1 incubator from Sartorius was used at a constant agitation rate of 120 rpm. Recombinant protein production used a standard procedure in which cultures were cooled from 37°C to 25°C when an optical density of 1.1 (± 10%) was reached. Isopropylthio-β-D-galactoside (IPTG) was added in a concentration of 0.25 or 1.0 mM, and the cultivation time after induction was 5 or 20 h. Cells were harvested by centrifugation and broken up with the lysis reagent B-Per . S. cerevisiae XR2μ was grown and processed as described recently [recently .A/min were measured. One unit of enzyme activity refers to 1 μmol of NAD(P)(H) consumed per minute. All measurements were performed with a Beckman DU-800 spectrophotometer using 50 mM potassium phosphate buffer, pH 7.5. The standard assay for CtXR contained 10 mM ethyl 4-cyanobenzoylformate and 250 μM NAD(P)H; that for CbFDH contained 200 mM sodium formate and 2 mM NAD+. Five % (v/v) ethanol was added to the buffer to enhance the solubility of ethyl 4-cyanobenzoylformate. Reactions were always started by the addition of coenzyme. Measured rates were corrected for appropriate blank readings accounting for non-specific oxidation or reduction of NAD(P)(H) by the cell extracts. Protein concentrations were determined with the BCA assay (Pierce) using bovine serum albumin as a standard. Determination of the activities of CtXR, ADH, and AlDH in the cell-free extract of S. cerevisiae was described previously [Reductase and dehydrogenase activities were assayed spectrophotometrically at 25°C, monitoring the reduction or oxidation of NAD(P)(H) at 340 nm over a time of 1–5 min. Typically, rates of 0.05 – 0.10 Δeviously .CDW/L E. coli XR_FDH and 100 mM ethyl 4-cyanobenzoylformate in 100 mM potassium phosphate buffer, pH 7.5, were incubated in 1.5 mL Eppendorf tubes at 30°C. Tubes were incubated for 0.5, 1, 2, 3 or 4 h. The reaction mixture was diluted 20-fold with buffer such that no organic phase (from insoluble substrate) remained, and cells were then collected by centrifugation. After cell lysis using B-Per, enzyme activities were assayed as described above.Reaction mixtures  containing 40 gCDW/L were suspended in 100 mM potassium phosphate buffer, pH 7.5. Ethyl 4-cyanobenzoylformate is a liquid and was added in a concentration between 10 and 500 mM as indicated. Because the solubility of ethyl 4-cyanobenzoylformate was only 10 mM under the conditions used, reactions with substrate concentrations of > 10 mM, took place in an aqueous-organic two-phase system. The concentration of sodium formate always exceeded that of the ketone substrate by 50 mM (minimum 150 mM). The total reaction volume was 1 mL, and conversions were started through addition of substrate. In reactions where a water-immiscible organic co-solvent  or ionic liquid (BMIMPF6) was used, the substrate was dissolved in the co-solvent first and added to the aqueous phase containing the cells in a 1:1 v/v ratio. A potassium phosphate buffer solution was used as control under otherwise identical conditions. 1 mL samples were taken at certain times, typically every hour, and analyzed as described under Analytical methods.Experiments were carried out at 30 (± 1) °C using 2-mL Eppendorf reaction tubes that were incubated in an end-over-end rotator (SB3 from Stuart) at 30 rpm. Cells in a concentration between 5 and 80 gS. cerevisiae XR2μ were described in a recent paper [Procedures used in whole-cell reductions catalyzed by nt paper .R and S antipode in a racemic mixture of ethyl 4-cyanomandelate was obtained when using acetonitrile and water  as eluent at a flow rate of 0.5 mL/min and a temperature of 40°C. Authentic (relevant) standards were used for peak identification, and quantification was based on peak area that was suitably calibrated with standards of known concentration. Reported yields of product on substrate consumed are always from analytical data because product isolation  was beyond the scope of this study.Samples were diluted with ethanol as required to obtain a homogeneous liquid phase. Cells were then separated by centrifugation. The supernatant was analyzed by chiral HPLC using a LaChrom HPLC system (Merck-Hitachi) equipped with a reversed phase CHIRALPAK AD-RH column from Daicel  and an L-7400 UV-detector. Detection was at 210 nm. Baseline separation of the The authors declare that they have no competing interests.RK has made substantial contributions to conception, design of experiments and acquisition of data. Carried out analysis, interpreted the data and has been involved in drafting the manuscript. MP contributed to acquisition of data. SE carried out the molecular genetic work. BN has made substantial contributions to conception, design of experiments, interpretation of data and has drafted the manuscript. All authors read and approved the final manuscript.


Adjuvant local-regional radiotherapy (LRRT) is routinely recommended for breast cancer patients. It is well known being related to pulmonary side-effects. We studied post-RT radiological changes on X-ray and CT, and correlated the findings with Quality of Life (QoL), common dosimetric factors and co-variates. The results were compared with a previously reported cohort of 137 irradiated women.20 < 30%. The lung field was divided into 3 regions and the development of post-RT density changes were graded (0-3). Patients with radiological changes were compared with non-responders. Clinical symptoms were registered and data on patient and treatment related co-variates were gathered prospectively. The ipsilateral lung dosimetric factors V13, V20, V30 and mean dose were calculated and QoL was assessed before and 4 months after RT.88 women underwent chest X-ray and CT pre-and 4-5 months after 3-D planned LRRT, minimizing the dose to the ipsilateral lung to V13 correlated independently with radiological changes on CT . The Co-variates smoking habits, age, chemotherapy, endocrine or trastuzumab therapy did not influence the outcome on multivariate analysis. QoL changes in physical function, i.e. fatigue, dyspnoea were not detected but there was a trend for a worse recovery after chemotherapy in patients with high V13 (Spearman Rank Correlation: p < 0.05).The use of dose-volume constraints significally reduced moderate-severe radiological changes on chest X-ray compared with our earlier study (Chi square trend test: p < 0.001). Symptomatic pneumonitis was also rare in the present study. No agreement was found between CT and chest X-ray as diagnostic tools for post-RT pneumonitis. V13 alone was correlated with occurrence of lung changes on CT.The use of dose-volume constraints significantly reduced post-RT radiological changes on chest X-ray in LRRT for BC. The lung changes on CT were also generally limited when we used this strategy and was not always picked up on chest X-ray. Variation in V The tg volume . We therg volume ,7 and tag volume  and RT-ig volume ,9. Indivg volume . Howeverg volume -13. In cg volume . Side-efg volume . Signs og volume ,17.20 < 30% was used and to correlate the findings with common dosimetric factors , Quality of life (QoL)-effects, symptoms and co-variates and compare the outcome to a previously reported study of 137 irradiated women [This study was performed to evaluate radiological pneumonitis (RP) on X-ray and CT in irradiated breast cancer women when the lung dose-volume constraints of Ved women .This study was approved by the local ethics committee. Participating women gave informed consent before study enrolment.All women who were referred to the Radiotherapy Department at Stockholm Söder Hospital during 2003-2005 for adjuvant LRRT after surgery for early breast cancer were asked to participate in this trial. Ninety-five patients were included, but seven patients withdrew their consent due to early relapse and were not evaluable. Eighty-eight patients were thus followed for seven months after RT for symptoms of acute/subacute radiation induced pulmonary complication. Mastectomy was done in 69 patients, while 19 patients were operated with conservative breast surgery. Seventy-two patients were irradiated with LRRT to the chest wall or breast, axilla and supraclavicular region and in these patients the internal mammary lymph nodes (IMN) were included. A total of 16 patients received RT excluding the IMN, i.e. 9 patients were given irradiation to the breast, axilla and supraclavicular region and 7 patients were referred for RT to the axilla and supraclavicular fossa only.The mean age of the patients was 56 years (range 32-81). Data on potential confounding factors were collected prospectively, i.e. history of cardio vascular or pulmonary co-morbidity, smoking habits, functional level (i.e. not being able to climb three flights of stairs without a rest due to shortness of breath) and adjuvant hormonal -, trastuzumab- and chemotherapy treatment.The chemotherapy was concluded 3-4 weeks prior to RT. Concurrent chemotherapy was never given. The typically regime consisted of doxorubicin, cyclophosphamide and 5-fluorouracil, but in 28 patients the therapy included docetaxel. Five patients received trastuzumab during RT. Intake of tamoxifen and anastrozol during RT was evenly split among the women.To asses and evaluate Quality of life (QoL) before and after RT we used the EORTC QLQ-C30 version 3.0 and the EORTC QLQ-BR23 questionnaires.13), >20 Gy (V20), >30 Gy (V30) and mean lung dose were defined.The used RT treatment techniques are described in detail in an earlier publication . LRRT afAll patients were followed for respiratory symptoms, i.e. cough, dyspnoea with or without fever, 1, 4 and 7 months after the termination of RT. The patients were classified into three groups according to CTC-criteria (version 2.0) .0. No complications: no registered respiratory symptoms monitored by the clinician.1. Mild reaction: cough and/or dyspnoea with our without fever judged to be radiation induced.2. Moderate reaction: same as 1 but with impaired daily functions and treated with corticosteroids.CT of the thorax was performed before and 4 months after RT and standardized chest X-ray was conducted after 5 months and evaluated by the same specialist in diagnostic radiology (HB) as in our previous trial [On the frontal chest radiograph the lung was divided into the three regions suggested by Arrigada, i.e. the apical-lateral (A-L), central-parahilar (C-P) and basal-lateral (B-L) regions . The borWe used the European Organisation for Research and Treatment of Cancer (EORTC) form QLQ-30 (version 3.0)  and the nd form. The form was double-sided and in some cases not completed on the back page. Scoring was performed in according with the EORTC scoring manual. Statistics and missing data were handled according to the manual. A four-point response scale was used to asses each item concerning functions or symptoms from 1  to 4 (very much), and a seven-point scale was used for global health status/QoL from 1 (very poor) to 7 (excellent). The scale scores were linearly transformed into scores of 0-100 according to the EORTC manual. A high score on the global health status/functional scale represents a high/healthy level of functioning. In contrast a high score on the symptom scale represents a high level of symptomatology/problems. A study of the subjective significance of changes in QoL scores has suggested that a mean change of 5 to 10 on the multi-item scales is perceived as little change, 10 to 20 as moderate change and greater than 20 as very much change. Greater than ten points on the transformed questionnaire scale were considered clinically meaningful [Eighty-one patients completed both measurements. In seven cases the pts did not receive the 2aningful ,23.In the present paper, three functional scales  and four symptom scales  were included from QLQ C-30. In the EORTC QLQ-BR23 form we included functional scales (future perspective).The relation between symptomatic and radiological RP and the relation between radiological RP and the dosimetric factors and co-variates was analyzed with univariate and multivariate logistic regression . Chi square trend test was used for test of correlation between radiological RP on X-ray in the present and earlier studies. To test agreement between CT-and X-ray for the diagnosis of radiological RP, Kappa-statistics was used.13 [13 were evaluated with Spearman Rank Correlation. All reported results were based on two-sided tests and p-values < 0.05 were considered statistically significant.Receiver operating characteristics curves (ROC) were used to predict radiological RP with V13 . ChangesFigure 20 for responders and non-responding patients are shown in Figure 20 and MLD in our previous study was 35% and 16 Gy, respectively [The use of dose-volume constraints significally reduced moderate-severe radiological RP on X-ray compared with the earlier treatment series for the technique with LRRT + IMN (Chi square trend test p < 0.001)   [13 was stronger correlated to MLD than V20.There was no agreement between X-ray and CT as diagnostic tools for post-radiological RP, (Kappa test) Table . V13 wasea: 0.7) . No otheMost of the side effects from RT appeared to have little effect on QoL in the present trial. Chemotherapy was concluded 3-4 weeks prior to RT and the patients started with a higher score on fatigue at baseline due to this.The variables role functioning, social functioning and future perspective, were improved 4 months after RT compared to baseline  there was thus a negative correlation. There was a significant correlation between high V13 and difficulties to take short walks, which could be of clinical significance, and the correlation was reported also when patients rated there overall total quality of life during the last week.Patients with high VRT Table . When ch20 to the ipsilateral lung to <30% was used for LRRT in early breast cancer. The result indicates that the used dose-volume constraints significantly reduced moderate-severe radiological RP on chest X-ray, in the present series, compared to our previous study [13) was correlated with occurrence of radiological RP on CT. ROC analyses was performed, yet the area under the curve was only 0.7 which is not an ideal predictive value [Clinically significant radiological and symptomatic RP was rare in this study when 3-D treatment planning, aiming at minimizing Vus study . The lunve value . Co-variThe LRRT-IMN group of our present series included only nine patients. We used the same RT-technique in both this and the previous study, and as could be expected, there was no difference in radiological RP Table . CTV volThe AI treatment in combination with RT is investigated in a randomised trial presented in Lancet Oncol. 2010. The results suggested that AI can be used early, but there still are doubts on potential long-term toxic effects, mainly cardiac in combination with RT ,27.Genetic factors may also play a vitale role in treatment. By identifying genetic factors associated with radiosensitivity it will be easier to predict which patients are at increased risk for complications secondary to radiation treatment -13.Even though symptomatic and radiological RP were rare in our trial, they still could be of importance if they prevail, as late changes could increase the risk of secondary lung cancer. This increased risk is seen in smokers five years after RT .To improve radiotherapy techniques and continue to study pulmonary morbidity and QoL after RT, is of great importance as breast cancer is a common disease among women.13 was most strongly related to radiological RP on CT. V13 was stronger correlated to MLD than V20, and may be an important metric in future trials on RT-induced lung toxicity.In conclusion, V20-constraints significantly reduced post-RT radiological changes on chest X-ray in LRRT for breast cancer. Symptomatic pneumonitis was, furthermore, rare in the present study when this strategy was used. There was no agreement between X-ray and CT as diagnostic tools for post-RT in this trial, as the lung changes typically were too limited for detection on X-ray. VThe authors declare that they have no competing interests.UBG coordinated the study, collected the data and drafted the manuscript.UBG, BW, GS and PL were involved with the design of the study.HB and GS analysed X-ray and CT diagnostics. BW analysed RT-doses. PL supported with the statistics. All authors read and approved the final manuscript.


Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor npr1 (nonexpressor of pathogenesis-related genes), and sid2 (SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six npr1 suppressors that accumulated lower levels of SA than npr1 upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified eds5 mutant. The other four are more susceptible than npr1 to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 and their identity merits further investigation.On the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, Acinetobacter sp. ADPWH_lux-based SA estimation, and allowed the screening for npr1 suppressors that accumulated less SA than npr1 after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.The rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor  Salicylic acid (SA) is a key signaling molecule in plant defense against biotrophic pathogens ,2. Upon SID1/EDS5 and SID2/EDS16, which were later shown to encode a chloroplast MATE (multidrug and toxin extrusion) transporter . For SAG, 10 units of β-glucosidase were added to the tube. After incubation at 37°C for 1.5 hr, an equal volume of 10% TCA was added to both tubes. After centrifugation at 14,000 g for 10 min, the supernatant was transferred to a fresh tube and partitioned with 1 mL extraction solvent (1 ethylacetate: 1 cyclohexane). The top organic phase was transferred to a new tube, and dried in a speed vacuum to final volume ~25 μL. The residue was resuspended to 0.25 mL with 0.2 M sodium acetate buffer (pH 5.5). After centrifugation at 14,000 g for 10 min, the supernatant was used for HPLC analysis. The sample was eluted with 0.2 M sodium acetate buffer pH 5.5 in 10% methanol at a flow-rate of 0.80 mL/min.An overnight culture of e et al. . BrieflyAll statistical analyses were performed with the data analysis tools  in Microsoft Excel of Microsoft Office 2004 for Macintosh.The authors declare that they have no competing interests.GM, RC, YD, DS, and XZ performed the experiments. ZM designed the project, wrote the manuscript, and is the PI of the laboratory. All authors read and approved the final manuscript.Detailed protocol for identification of SA metabolic mutants using the direct boiling method.Click here for file


Reemerging bluetongue virus serotype 8 (BTV-8) in Germany was detected first in May 2007 in a sentinel cow and in February 2008 in an export heifer. Reemergence was confirmed by retesting the samples, experimental inoculation, fingerprinting analysis, and virus isolation. Overwintering of BTV-8 and continuous low-level infections are assumed. Orbivirus. BTV is transmitted to its hosts by the bite of Culicoides spp. midges. It causes a noncontagious, arthropod-borne disease of domestic and wild ruminants and camelids (Bluetongue virus (BTV) is a double-stranded RNA virus of the genus To detect reemerging BTV-8 in Germany, a sentinel animal program was started in March 2007. Each month, the program tested 150 BTV-naive cattle per 2,025 km². Screening was performed by ≈30 regional laboratories located in the 16 federal states of Germany. Diagnostic testing was performed with commercially available BTV-blocking-ELISAs  and validated BTV real-time PCRs  tested positive for BTV antibodies and genomes in a regional laboratory . This animal had tested negative for BTV on March 3 and April 3. After the positive test result, the original samples as well as new samples were transferred to the reference laboratory for further investigation. The sample collected on May 3 and all further samples of this animal tested positive by BTV ELISA as well as by BTV real-time reverse transcription–PCR (RT-PCR). According to Deutscher Wetterdienst, available from A). Therefore, new BTV-infection of a BTV-naive animal was suggested. To confirm the presence of infectious BTV, fresh EDTA-blood samples from the positive animal were experimentally injected into 2 bovids (40 mL intravenously and 10 mL subcutaneously). To mimic natural exposure by the bite of a midge, only 20 µL was injected subcutaneously into a 3rd bovid (B). Testing of 80 contact animals from the same herd showed 38 antibody-positive cattle but no additional RT-PCR–positive case.Samples taken on May 3 had positive ELISA titers of 8–16, and the cycle threshold (Ct) values of the real-time RT-PCR analysis were 25.6–27.9 , panel Ard bovid . Both anrd bovid . Furtherrd bovid , panel BIn February 2008, a 2-year-old Holstein Friesian heifer, destined for export from Schleswig-Holstein, tested positive for BTV antibodies and genomes during quarantine. Testing was done by a regional laboratory . Whereas a first blood sample collected on January 22, 2008, was negative according to real-time RT-PCR; a subsequent sample taken on February 15 was positive by real-time RT-PCR and had a Ct value of 25.13. Both test results were confirmed by the German National Reference Laboratory for BTV. For the positive EDTA blood sample, a Ct value of 25.1 and an ELISA inhibition value of 85.7% (positive >50%) were ascertained. Furthermore, fingerprinting analysis  confirmed that both the negative and the positive samples were from the same animal (data not shown). The meteorologic data from January 20 to February 15 showed unsuitable weather conditions for BTV replication in outdoor midges at the time of BTV transmission; daily minimum temperatures were 7.2°C to –6.3°C, and daily maximum temperatures 3.4°C–13.4°C. Finally, BTV was isolated after inoculation of Vero cell cultures with the real-time PCR-positive blood samples.According to the dates of sampling and subsequent seroconversion, genome detection, and Ct values, 1 animal became infected during April 1–25, 2007, and the other became infected during January 20–February 12, 2008. According to meteorologic data, infection between April 15 and 25 is likely, whereas the suggested infection time in 2008 was not associated with weather suitable for BTV transmission. Therefore, we have drawn several conclusions. First, BTV-8 reemerged in Europe in 2007 and 2008 unexpectedly early. This timing makes it unlikely that a real vector-free period exists in northwest Europe. Second, the detection of both cases was fortuitous. Because of the low number of sentinel animals in 2007, or double-tested cattle destined for export in January and February 2008, substantially more new infections may have occurred in winter 2007 and early spring 2008. Therefore, overwintering of BTV-8 appears to be efficient, and under the climatic conditions of winter and spring 2007 and 2008 in Germany, continuous infections occurring at a low level must be assumed. However, the detailed mechanisms of overwintering remain unclear. In addition, the meterologic data connected to the early case in 2008 suggest indoor survival of infected midges. Third, sentinel animals proved to be useful for the early detection of recirculating virus. However, establishing and conducting an efficient sentinel program are complex and expensive endeavors, and, as shown in 2008, positive cases might be detected earlier by chance.On the basis of our experience, we recommend that all samples that could represent new infections in a given region or season should be confirmed by a reference laboratory that as a minimum standard confirms identity of the origin of all samples. This step would avoid misleading interpretations due to, for example, a mix-up of samples or animals. Also, depending on future climate in the BTV-8–infected regions in 2008, the first BTV-8 case could indicate the need for early protection, especially of sheep and goats, by BTV-8–specific vaccination.